Ever since the inception of recombinant DNA technology, laboratory and preparative expression of heterologous proteins in Escherichia coli has been a cornerstone of the biotechnology enterprise. Baneyx, F. & Mujacic, M. Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22, 1399-1408 (2004); Swartz, J. R. Advances in Escherichia coli production of therapeutic proteins. Curr. Opin. Biotechnol 12, 195-201 (2001); Georgiou, G. & Segatori, L. Preparative expression of secreted proteins in bacteria: status report and future prospects. Curr Opin Biotechnol 16, 538-545 (2005). Unfortunately, many eukaryotic proteins expressed in the cytoplasm of E. coli are prone to misfolding and are subsequently degraded by cellular proteases or deposited into biologically inactive aggregates known as inclusion bodies. Dyson, M. R., Shadbolt, S. P., Vincent, K. J., Perera, R. L. & McCafferty, J. Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC Biotechnol 4, 32 (2004); Luan, C. H. et al. High-throughput expression of C. elegans proteins. Genome Res 14, 2102-2110 (2004); Braun, P. et al. Proteome-scale purification of human proteins from bacteria. Proc Natl Acad Sci USA 99, 2654-2659 (2002); Baker, T. A. & Sauer, R. T. ATP-dependent proteases of bacteria: recognition logic and operating principles. Trends Biochem Sci 31, 647-653 (2006); Tomoyasu, T., Mogk, A., Langen, H., Goloubinoff, P. & Bukau, B. Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol. Mol Microbiol 40, 397-413 (2001); Bowden, G. A., Paredes, A. M. & Georgiou, G. Structure and morphology of protein inclusion bodies in Escherichia coli. Biotechnology (N Y) 9, 725-730 (1991); Villayerde, A. & Carrio, M. M. Protein aggregation in recombinant bacteria: biological role of inclusion bodies. Biotechnol Lett 25, 1385-1395 (2003).
Misfolding of eukaryotic proteins in the cytoplasm is often a consequence of the relative crowdedness of this compartment where macromolecule concentration can reach 300-400 mg/ml and the requirement for post-translational processing that is absent from the cytoplasm such as disulfide bond formation or glycosylation. Ellis, R. J. & Minton, A. P. Cell biology: join the crowd. Nature 425, 27-28 (2003); Kadokura, H., Katzen, F. & Beckwith, J. Protein disulfide bond formation in prokaryotes. Annu Rev Biochem 72, 111-135 (2003); Weerapana, E. & Imperiali, B. Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems. Glycobiology 16, 91R-101R (2006).
To remedy some of these issues, secretion into the periplasm of E. coli is often employed because this compartment: (1) contains significantly fewer proteins, especially proteases, compared to the cytoplasm; consequently periplasmic proteins are easier to isolate and are often less prone to crowding-induced aggregation and/or proteolytic degradation; and (2) houses a network of redox enzymes that catalyze the formation and isomerization of disulfide bonds that are essential for the folding and function of many eukaryotic proteins. These advantages notwithstanding, there still remain significant challenges with respect to secretion across the inner membrane, degradation by resident periplasmic proteases and misfolding due to either incorrect disulfide-bond formation or aggregation into periplasmic inclusion bodies. Thus, to address these challenges, new experimental tools are needed for understanding and characterizing the complexities of the periplasmic folding environment and elucidating the factors that impede the folding of proteins in this biological compartment.